我们感谢万实验室和Nagarajan实验室的成员进行了详细的讨论。 N.Nagarajan得到A * STAR的资助。姚先生获得了A * STAR和Science-Branco Weiss奖学金社会的资助。

1.用生物素化补骨脂素和RNA提取处理HeLa细胞<ol><li>在10cm板中补充有10％胎牛血清（FBS）和1％青霉素链霉素（PS）的Dulbecco改良的Eagle培养基（DMEM）中培养HeLa细胞。 </li><li>用5毫升1x PBS洗涤HeLa细胞两次。通过将盘垂直放置1分钟，将多余的PBS完全从盘中排出。 </li><li>向细胞中均匀加入含有200μM生物素化补骨脂素和0.01％w / v卵磷脂的1mL PBS，并在37℃下孵育5分钟。虽然生物素化的补骨脂素可以进入细胞，当细胞暴露于少量的洋甙素（0.01％）5分钟时，其渗透性要高得多</li><li>取出含有处理过的HeLa细胞的10厘米平板的盖子，将盘子平放在冰上。使用UV交联剂，将含有细胞的培养皿用365nm紫外线照射20分钟，距离UV灯泡3厘米的距离。 </li><li>隔离t根据制造商的方案，通过硫氰酸胍 - 苯酚 - 氯仿提取来自HeLa细胞的RNA。加入1体积的异丙醇以在室温下沉淀RNA 30分钟。 暂停点:RNA可以无限期地在-20°C储存在异丙醇中。可以在该步骤中进行斑点印迹以检查生物素标记的补骨脂素是否已经成功进入细胞并且在体内具有交联的RNA（ 图2 ）。 </li><li>在4℃下以20,000×g离心沉淀的RNA 30分钟以回收RNA。去除上清液，并加入1 mL 70％冷乙醇至RNA颗粒中以洗涤RNA。 </li><li>在4℃下将样品以20,000×g离心15分钟。取出上清液，并在室温下空气干燥沉淀5分钟。 </li><li>向RNA沉淀中加入无核酸酶的水，并使用紫外光谱仪定量RNA的浓度。 暂停点:f。可在-80°C储存RNA睫毛在液氮中冻结。 </li></ol> RNA分裂<ol><li>在0.2 mL PCR管中预热15μL2x RNA片段化缓冲液和10μLRNA样品（1μg/μL），95°C 1 min。通过上下移液将10μL加热的2X RNA片段缓冲液与10μLRNA样品混合。在95℃孵育样品5分钟。通过在冰上冷却反应停止反应。 </li><li>将2个碎裂反应转移并汇集到1.5 mL离心管中。向反应液中加入40μL无核酸酶的水，10μL3 M乙酸钠，1μL糖原和300μL100％乙醇进行裂解反应。将RNA在-20℃孵育至少1小时以沉淀RNA。 </li><li>将沉淀的RNA以20,000×g离心30分钟，除去上清液并用1mL 70％乙醇洗涤RNA。 </li><li>将RNA以20,000 xg离心15分钟，上清，并将RNA溶解在20μL无核酸酶的水中。 </li></ol> RNA大小选择和洗脱<ol><li>向微量离心管中回收的RNA中加入20μLRNA加载染料。用RNA加载染料在95℃加热RNA 3分钟，并将其置于冰上2分钟。 </li><li>在微量离心管中加入3μLRNA加载染料至0.25μL10 nt DNA梯。将梯子在95℃下加热3分钟，并将其放在冰上2分钟。 </li><li>将变性的梯子和样品加载到8.6厘米x 6.8厘米6厘米的TBE尿素凝胶上，并在180℃下通过电泳电泳40分钟。将凝胶在10mL含有1:10,000核酸凝胶染色剂的TBE缓冲液中染色5分钟在黑暗中。 </li><li>使用24 G针刺穿底部的干净的0.6 mL微量离心管，并将其置于2 mL微量离心管中。 </li><li>使用透照器观察后染色凝胶上的条带，并切割对应于90-11的凝胶切片0点将凝胶切片转移到2 mL微量离心管中穿刺的0.6 mL微量离心管中。执行该大小选择以允许嵌合片段具有最小长度以映射到转录组，并在下游尺寸选择步骤中区分接近连接的产物与未产生的产物。 </li><li>将凝胶切片在室温下以12,000xg离心2分钟，以切碎凝胶切片并将其收集在2mL微量离心管中。丢弃空的0.6 mL离心管。向2 mL微量离心管中的切碎的凝胶切片中加入700μL洗脱缓冲液，并在4℃下恒温孵育过夜，以使样品扩散到缓冲液中。 </li><li>将凝胶切片和洗脱缓冲液转移到离心管过滤器中，并以20,000 xg离心2分钟。凝胶片将被捕获在过滤器的顶部隔间。丢弃凝胶片和顶部隔间。 </li><li>加入1μL糖原，7将100μL异丙醇加入到微量离心管中的滤液中，并将RNA在-20℃下沉淀至少1小时或过夜。 暂停点:RNA可以无限期地在-20℃下在异丙醇中沉淀。 </li><li>在4℃下以20,000×g离心沉淀的RNA 30分钟以回收RNA。取出上清液，加入1 mL 70％冷乙醇洗涤RNA沉淀。在4℃下以20,000xg离心洗涤的沉淀15分钟。 </li><li>取出洗涤缓冲液，加入20μL无核酸酶的水重新悬浮RNA。使用UV光谱仪定量RNA的浓度。 暂停点:RNA可以在-80°C储存，在液氮中快速冷冻。 </li></ol> 4. RNA交联区域的丰富<ol><li>涡旋链霉抗生物素蛋白包被的磁珠强力地将细珠重新悬浮在管中。将100μL珠子转移到1.5 mL离心管中，并将其放在磁铁上ic放置1分钟以使珠子粘附到管的磁性侧。小心地从小珠中取出储存缓冲液，并将管从磁铁架上取出。 </li><li>向微量离心管中的珠子中加入1 mL裂解缓冲液，上下移液。将含有微珠的微量离心机放置在磁性架上1分钟，以将珠子与洗涤缓冲液分离。重复这一次总共3次洗涤。 </li><li>通过将含有微珠的微量离心机放置在磁性架上1分钟，将洗涤缓冲液从珠粒中取出。将RNA酶抑制剂（1:200）加入裂解缓冲液中，并将100μL裂解缓冲液加入到微量离心管中洗涤的珠粒上。 </li><li>加入2mL新鲜制备的杂交缓冲液，1mL补充的裂解缓冲液，1.5μg大小分级的RNA和100​​μL重悬珠珠至15mL锥形离心管中。轻轻旋转管。 </li><li>将15 mL锥形离心管在37°C下孵育30分钟，端对端旋转。在37°C预洗洗涤缓冲液。 </li><li>将15 mL锥形离心管放在磁性支架上15 mL管中2 min，将珠子与缓冲液分离。将缓冲液从管中小心取出并将其丢弃。 </li><li>将1mL预热的洗涤缓冲液加入到珠中，上下移液并将珠转移到1.5mL的微量离心管中。在37℃下，在珠子上孵育5分钟，进行端对端旋转。 </li><li>简单地离心微量离心管的内容物。将微量离心管中的1.5 mL洗涤珠置于磁性支架上2 mL试管中1分钟，以将珠子与洗涤缓冲液分离。从微量离心管中轻轻移取缓冲液，从珠中取出缓冲液。 </li><li>将1mL预热的洗涤缓冲液加入到珠子中并向上和向下移液以重复洗涤。共执行5次洗涤。在第五次洗涤结束时，通过将含有微珠的微量离心机放置在磁体架上来除去洗涤缓冲液1分钟。 </li></ol>邻近连接<ol><li>将1 mL冷T4多核苷酸激酶（PNK）缓冲液加入到洗涤的珠中，并在4℃下孵育珠子5分钟，并以端对端旋转。将含有珠粒的微量离心管置于磁条上1分钟，轻轻取出T4 PNK缓冲液。重复此步骤总共2次洗涤，并在最后一次洗涤后取出T4 PNK缓冲液。 </li><li>根据表1将缓冲液加入珠中。为了使RNA片段的3&#39;末端能够连接到下面的3&#39;衔接子上，将缓冲液中的珠子在37℃下恒温搅拌4小时，将RNA末端的3&#39;环状磷酸酯基团转化为3&#39;OH。 </li><li>根据表2添加缓冲液到前面的反应。为了使RNA片段的5&#39;末端具有连接能力，在37℃下在ATP存在下恒定搅拌下孵育反应1小时将RNA上的5&#39;OH转化为5&#39;磷酸。 </li><li>根据表3 ，将缓冲液加入前一反应进行接近连接。在16℃下反应16h恒温搅拌， </li><li>将含有连接的RNA的微量离心机置于磁体架上1分钟。小心取出上清液，并加入1 mL室温洗涤缓冲液至珠粒。通过上下移动重悬。 </li><li>将微量离心管放置在磁体架上1分钟，并小心地取出洗涤缓冲液。执行总共2次洗涤，并在第二次洗涤结束时从珠中除去洗涤缓冲液。 </li><li>加入100μL的RNA PK缓冲液加入珠中，并通过上下移液重悬。将样品在95℃加热10分钟。 </li><li>将样品在冰上冷却1分钟，并向样品中加入500μL硫氰酸胍 - 苯酚 - 氯仿。通过剧烈涡旋混合10秒。孵化mixtu在室温下放置10分钟。 暂停点:RNA可以在-80°C下储存在硫氰酸胍 - 苯酚 - 氯仿中几个月。 </li><li>向硫氰酸胍 - 苯酚 - 氯仿提取样品中加入100μL氯仿。大力涡旋10秒。在4℃下将样品以20,000×g离心15分钟。 </li><li>将400μL水层转移到新的1.5 mL微量离心管中。加入800μL（2体积）100％乙醇，并通过上下移液混合。 </li><li>将RNA溶液转移到RNA清除柱上，按照制造商的说明回收RNA，确保即使保留了小RNA。在100μL无核酸酶的水中洗脱RNA。 暂停点:RNA可以在-80°C储存，在液氮中快速冷冻。 </li></ol> 6.生物素化补骨脂素的反向交联<ol><li>将100μL洗脱的样品转移到24孔板的孔中吃了。取下盖子并在UV 254nm下照射样品5分钟。 </li><li>将反向交联的样品转移到干净的微量离心管中。加入10μL乙酸钠，1μL糖原和300μL100％乙醇，并将RNA在-20℃下沉淀至少1小时或过夜。 暂停点:RNA可以无限期地在-20℃下在乙醇中沉淀。 </li><li>在4℃下以20,000×g离心沉淀的RNA 30分钟以回收RNA。取出上清液，加入1 mL 70％冷乙醇洗涤RNA沉淀。 </li><li>在4℃下以20,000xg离心洗涤的沉淀15分钟。取出洗涤缓冲液，加入4.25μL无核酸酶的水重新悬浮RNA。将RNA转移到0.2 mL PCR管中。 暂停点:RNA可以在-80°C储存，在液氮中快速冷冻。 </li></ol>反转录和cDNA循环<ol><li>加入0.75μL的RNA接头（0.5μg/μL）重悬于PCR管中，并在80℃加热90秒。将样品放在冰上2分钟。 </li><li>根据表4 ，将缓冲液添加到3&#39;接头连接的变性样品中。在25℃下孵育反应2.5小时。 </li><li>将反应转移到1.5 mL离心管中。向反应液中加入90μL无核酸酶的水，然后加入10μL乙酸钠，1μL糖原和300μL100％乙醇。将RNA在-20℃沉淀至少1小时或过夜。 暂停点:RNA可以无限期地在-20℃下在乙醇中沉淀。 </li><li>在4℃下以20,000×g离心沉淀的RNA 30分钟以回收RNA。取出上清液，加入1 mL 70％冷乙醇洗涤RNA沉淀。 </li><li>在4℃下以20,000g离心洗涤的沉淀15分钟。取出洗涤缓冲液，将沉淀物重新悬浮在5μL无核酸酶的水溶液中亚特。 </li><li>向微量离心管中回收的RNA中加入5μLRNA加载染料。用RNA加载染料在95℃加热RNA 3分钟，并将其置于冰上2分钟。 </li><li>在微量离心管中加入3μLRNA加载染料至0.25μL10 nt DNA梯。将梯子在95℃下加热3分钟，并将其放在冰上2分钟。 </li><li>使用8.6 cm x 6.8 cm 6％TBE尿素凝胶进行尺寸分级，如步骤3.3和3.4所示。 </li><li>使用透照器显现用核酸染色的凝胶，并在梯子上切下对应于110-140nt的凝胶切片。将凝胶切片转移到2 mL微量离心管中穿刺的0.6 mL微量离心管中，并按照步骤3.6 - 3.9的方案进行。 </li><li>加入5μL无核酸酶的水重悬RNA颗粒。将RNA转移到0.2 mL PCR管中。 暂停点:RNA可以在-80°C储存，在液氮中快速冷冻。 </li><li>在1.25加入1μLRT引物81; M至PCR管中的RNA，并在80℃加热2分钟。将样品放在冰上2分钟。 </li><li>根据表5添加反转录缓冲液。在50℃下反应30分钟。 </li><li>向反应中加入1.1μL1N NaOH，在98℃加热20分钟。将反应放在冰上。 </li><li>将反应转移到1.5 mL离心管中。向反应液中加入90μL无核酸酶的水，然后加入10μL乙酸钠，1μL糖原和300μL100％乙醇。将DNA在-20℃沉淀至少1小时或过夜。 暂停点:DNA可以无限期地在-20℃下在乙醇中沉淀。 </li><li>在4℃下将沉淀的DNA以20,000g离心30分钟以回收DNA。取出上清液，加入1 mL 70％冷乙醇洗涤DNA沉淀。在4℃下以20,000xg离心洗涤的沉淀15分钟。取出洗涤缓冲液重悬放入5μL无核酸酶的水。 </li><li>使用8.6 cm x 6.8 cm 6％TBE尿素凝胶进行尺寸分级，如步骤3.3和3.4所示。 </li><li>使用透照器显现后染色凝胶，并在梯子上切下对应于200-240nt的凝胶切片。由于加入96碱基RT引物，逆转录的cDNA现在比RNA片段长96个碱基。将凝胶切片转移到2 mL微量离心管中穿刺的0.6 mL微量离心管中。 </li><li>将凝胶切片在室温下以12,000xg离心2分钟，以切碎凝胶切片并将其收集在2mL微量离心管中。丢弃空的0.6 mL离心管。 </li><li>加入700μL洗脱缓冲液至2 mL离心管中切碎的凝胶切片，37°C恒温恒温孵育2 h。按照步骤3.7-3.8中所述的协议。 </li><li>加入6μL无核酸酶的水重新悬浮DNA沉淀。将DNA转移到0.2 mL PCR管中。 <li>将缓冲液加入到用于cDNA环化的反应中，根据表6 。将反应在60℃下孵育1小时，并在80℃下加热10分钟以使反应失活。 </li><li>将反应转移到1.5 mL离心管中。根据制造商的说明，使用DNA清除柱纯化环化的cDNA。向柱中加入20μL无核酸酶的水洗脱纯化的cDNA。 暂停点:DNA可以在-20°C储存几个月。 </li></ol> 8. PCR扩增（小规模PCR） <ol><li>将缓冲液加入根据表7的反应进行PCR扩增，以测试文库扩增所需的最小循环次数。 </li><li>根据表8设置PCR循环条件。暂停反应并转移5μL将PCR反应在10,15和20个循环中分离成分离的1.5mL离心管。 </li><li> 在不同周期的5μLPCR反应中加入1μL6x DNA凝胶加载染料。在3V琼脂糖凝胶电泳上以120V进行凝胶电泳1小时以显现PCR产物。 <ol><li>使用最低数量的PCR周期（Y），其显示在约250个碱基处的扩增（ 图3中 ，在15个循环的扩增周期和10个周期的微弱条带中存在强带），12个大规模PCR扩增循环可能生成足够的深度测序材料，因为使用的cDNA量是小规模PCR扩增的10倍）。 </li></ol></li></ol> 9. PCR扩增（大规模PCR）和纯化<ol><li>将缓冲液添加到根据表9的反应中进行大规模PCR的PCR扩增。 </li><li>根据PCR设置PCR循环条件表10 。 </li><li>将5μL6x DNA凝胶加载染料加入到25μLPCR反应中，并加载到3％琼脂糖凝胶的孔中。将1 kb加DNA梯子加入一个单独的孔。在100 V下进行凝胶电泳1.5 h。 </li><li>使用UV透射仪可视化完成的凝胶。 </li><li>提取含200-300个碱基的PCR产物的凝胶切片，并将凝胶转移到干净的2mL离心管中。 </li><li>从DNA凝胶提取试剂盒中加入1 mL凝胶溶解度缓冲液至凝胶切片，并在室温下孵育30分钟，或直到凝胶溶解，不断搅拌。 </li><li>向溶解的凝胶中加入200μL异丙醇，并通过移液进行良好的混合。将700μL样品转移到DNA凝胶提取清洗柱中，以13,000 xg离心30秒。继续将溶解的样品转移到色谱柱中，直到所有样品都装载到色谱柱上。 </li><li>加入500μL凝胶溶解度缓冲液，然后将700μL柱洗涤缓冲液加入柱中，根据制造商的方案。将12μL无核酸酶的水加入到色谱柱的中心以排除DNA。暂停点:DNA可以保存在-20°C。 </li><li>使用荧光定量法定量DNA的浓度。如果构建多个库并将其汇总在一起进行测序，则将相同量的每个样品添加到池中，以进行高通量测序。 </li></ol>

<ol><li>Wan, Y., Kertesz, M., Spitale, R. C., Segal, E., Chang, H. Y. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Understanding+the+transcriptome+through+RNA+structure%5BTitle%5D)+AND+%22Nat.Rev.Genet%22%5BJournal%5D)">Understanding the transcriptome through RNA structure.</a> Nat.Rev.Genet. 12 (9), 641-655 (2011).</li>
<li>Kertesz, M., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Genome-wide+measurement+of+RNA+secondary+structure+in+yeast%5BTitle%5D)+AND+%22Nature%22%5BJournal%5D)">Genome-wide measurement of RNA secondary structure in yeast.</a> Nature. 467 (7311), 103-107 (2010).</li>
<li>Wan, Y., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Genome-wide+Measurement+of+RNA+Folding+Energies%5BTitle%5D)+AND+%22Mol.Cell%22%5BJournal%5D)">Genome-wide Measurement of RNA Folding Energies.</a> Mol.Cell. 48 (2), 169-181 (2012).</li>
<li>Wan, Y., Qu, K., Ouyang, Z., Chang, H. Y. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Genome-wide+mapping+of+RNA+structure+using+nuclease+digestion+and+high-throughput+sequencing%5BTitle%5D)+AND+%22Nat.Protoc%22%5BJournal%5D)">Genome-wide mapping of RNA structure using nuclease digestion and high-throughput sequencing.</a> Nat.Protoc. 8 (5), 849-869 (2013).</li>
<li>Wan, Y., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Landscape+and+variation+of+RNA+secondary+structure+across+the+human+transcriptome%5BTitle%5D)+AND+%22Nature%22%5BJournal%5D)">Landscape and variation of RNA secondary structure across the human transcriptome.</a> Nature. 505 (7485), 706-709 (2014).</li>
<li>Spitale, R. C., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Structural+imprints+in+vivo+decode+RNA+regulatory+mechanisms%5BTitle%5D)+AND+%22Nature%22%5BJournal%5D)">Structural imprints in vivo decode RNA regulatory mechanisms.</a> Nature. 519 (7544), 486-490 (2015).</li>
<li>Ding, Y., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((In+vivo+genome-wide+profiling+of+RNA+secondary+structure+reveals+novel+regulatory+features%5BTitle%5D)+AND+%22Nature%22%5BJournal%5D)">In vivo genome-wide profiling of RNA secondary structure reveals novel regulatory features.</a> Nature. 505 (7485), 696-700 (2014).</li>
<li>Rouskin, S., Zubradt, M., Washietl, S., Kellis, M., Weissman, J. S. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Genome-wide+probing+of+RNA+structure+reveals+active+unfolding+of+mRNA+structures+in+vivo%5BTitle%5D)+AND+%22Nature%22%5BJournal%5D)">Genome-wide probing of RNA structure reveals active unfolding of mRNA structures in vivo.</a> Nature. 505 (7485), 701-705 (2014).</li>
<li>Kudla, G., Granneman, S., Hahn, D., Beggs, J. D., Tollervey, D. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Cross-linking%2C+ligation%2C+and+sequencing+of+hybrids+reveals+RNA-RNA+interactions+in+yeast%5BTitle%5D)+AND+%22Proc.Natl.Acad.Sci.U.S.A%22%5BJournal%5D)">Cross-linking, ligation, and sequencing of hybrids reveals RNA-RNA interactions in yeast.</a> Proc.Natl.Acad.Sci.U.S.A. 108 (24), 10010-10015 (2011).</li>
<li>Sugimoto, Y., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((hiCLIP+reveals+the+in+vivo+atlas+of+mRNA+secondary+structures+recognized+by+Staufen+1%5BTitle%5D)+AND+%22Nature%22%5BJournal%5D)">hiCLIP reveals the in vivo atlas of mRNA secondary structures recognized by Staufen 1.</a> Nature. 519 (7544), 491-494 (2015).</li>
<li>Engreitz, J. M., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((RNA-RNA+interactions+enable+specific+targeting+of+noncoding+RNAs+to+nascent+Pre-mRNAs+and+chromatin+sites%5BTitle%5D)+AND+%22Cell%22%5BJournal%5D)">RNA-RNA interactions enable specific targeting of noncoding RNAs to nascent Pre-mRNAs and chromatin sites.</a> Cell. 159 (1), 188-199 (2014).</li>
<li>Ramani, V., Qiu, R., Shendure, J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((High-throughput+determination+of+RNA+structure+by+proximity+ligation%5BTitle%5D)+AND+%22Nat.Biotechnol%22%5BJournal%5D)">High-throughput determination of RNA structure by proximity ligation.</a> Nat.Biotechnol. , (2015).</li>
<li>Lu, Z., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((RNA+Duplex+Map+in+Living+Cells+Reveals+Higher-Order+Transcriptome+Structure%5BTitle%5D)+AND+%22Cell%22%5BJournal%5D)">RNA Duplex Map in Living Cells Reveals Higher-Order Transcriptome Structure.</a> Cell. 165 (5), 1267-1279 (2016).</li>
<li>Sharma, E., Sterne-Weiler, T., O'Hanlon, D., Blencowe, B. J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Global+Mapping+of+Human+RNA-RNA+Interactions%5BTitle%5D)+AND+%22Mol.Cell%22%5BJournal%5D)">Global Mapping of Human RNA-RNA Interactions.</a> Mol.Cell. 62 (4), 618-626 (2016).</li>
<li>Aw, J. G., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((In+Vivo+Mapping+of+Eukaryotic+RNA+Interactomes+Reveals+Principles+of+Higher-Order+Organization+and+Regulation%5BTitle%5D)+AND+%22Mol.Cell%22%5BJournal%5D)">In Vivo Mapping of Eukaryotic RNA Interactomes Reveals Principles of Higher-Order Organization and Regulation.</a> Mol.Cell. 62 (4), 603-617 (2016).</li>
</ol>

作者没有竞争的经济利益。

在这里，我们详细描述了SPLASH的实验和计算工作流程，这是一种允许我们以全基因组方式识别成对RNA相互作用的方法。我们已经在细菌，酵母和人类文化中成功地利用SPLASH，并且预计该策略可以广泛应用于不同细胞状态下的多种生物。协议中的关键步骤之一是从至少20μg的交联RNA开始，为下游过程提供足够的材料。然后将RNA片段化至100个碱基并进行PAGE大小选择。这些步骤对于我们在图书馆生成过?...

研究大分子如何折叠和相互作用是理解细胞中基因调控的关键。在过去十年中，为了了解DNA和蛋白质如何促进基因调控，在过去十年中已经做了很多努力，对基因表达的转录后调控相对较少。 RNA在其线性序列及其二级和三级结构中携带信息1 。与其自身和其他物质配对的能力对于其在体内的功能是重要的。高通量RNA二级结构探测的最新进展已经为转录组2,3,4,5,6,7,8中的双链和单链区域的位置提供了宝贵的见解关于配对互动伙伴关系的信息仍然很大程度上缺失。为了确定哪个RNA序列与转录组中的另一个RNA区域相互作用，我们需要全球成对的信息。 以全球，公正的方式映射成对的RNA相互作用传统上是一个重大的挑战。虽然以前的方法，如CLASH 9 ，hiCLIP 10和RAP 11被用来以大规模的方式鉴定RNA相互作用，但是这些技术通常将RNA碱基配对映射到与特定蛋白质或RNA物种相互作用的RNA亚类。研究全球RNA相互作用的最新进展包括方法RPL 12 ，其不能在体内稳定RNA相互作用，因此可能仅捕获体内相互作用的子集。为了克服这些挑战，我们和其他人开发了全基因组，无偏见的策略p RNA 在体内相互作用，使用修饰版本的交联剂补骨脂素13,14,15 。在本协议中，我们描述了使用生物素化的补骨脂素在体内交联碱基配对RNA，然后进行邻近连接和高通量测序的P soralen交联，Ligigated和S选择Hyrbrids（SPLASH）的S等位基因的细节。识别RNA基因配对伙伴全基因组（ 图1 ） 15 。 在本手稿中，我们描述了使用培养的贴壁细胞进行SPLASH的步骤，在这种情况下是HeLa细胞。相同的方案可以容易地适应于悬浮哺乳动物细胞和酵母和细菌细胞。简言之，HeLa细胞用生物素化的补骨脂素处理并在365nm下照射以在体内交联相互作用的RNA碱基对。然后从细胞中提取RNA，用链霉抗生物素蛋白珠片段碎片化并富集交联区域。然后使用接近连接将相互作用的RNA片段连接在一起，并制成用于深度测序的cDNA文库。测序后，将嵌合RNA映射到转录组/基因组上以鉴定彼此配对的RNA相互作用区域。我们已经成功地利用SPLASH在酵母和不同人类细胞中识别数千个RNA相互作用，包括分子内和分子间RNA碱基配对在不同类别的RNA，如snoRNA，lncRNA和mRNAs，以瞥见结构组织和相互作用模式的细胞中的RNA。

<table><tbody><tr><td>1 kb Plus DNA Ladder</td><td>Life Technologies Holdings Pte Ltd</td><td>10787026</td><td>DNA ladder</td></tr><tr><td>10 bp DNA ladder</td><td>Life Technologies Holdings Pte Ltd</td><td>10821-015</td><td>DNA ladder</td></tr><tr><td>20% SDS solution</td><td>First BASE</td><td>BUF-2052-1L&amp;nbsp;&amp;nbsp;</td><td></td></tr><tr><td>20x SSC</td><td>First BASE</td><td>BUF-3050-20X1L</td><td></td></tr><tr><td>3.0 M Sodium Acetate Solution</td><td>First BASE</td><td>BUF-1151-1L-pH5.2&amp;nbsp;&amp;nbsp;</td><td>Required for nucleic acid precipitation</td></tr><tr><td>40% Acrylamide/Bis Solution, 19:1</td><td>Bio-Rad</td><td>1610145</td><td>TBE Urea gel component</td></tr><tr><td>Ambion Buffer Kit</td><td>Life Technologies Holdings Pte Ltd</td><td>AM9010</td><td></td></tr><tr><td>Ammonium Persulfate, Molecular Grade&amp;nbsp;&amp;nbsp;</td><td>Promega</td><td>V3131&amp;nbsp;</td><td>TBE Urea gel component</td></tr><tr><td>Bromophenol Blue</td><td>Sigma-Aldrich</td><td>B0126-25G</td><td></td></tr><tr><td>Chloroform</td><td>Merck</td><td>1.02445.1000</td><td>RNA extraction</td></tr><tr><td>Single strand DNA ligase</td><td>Epicentre</td><td>CL9025K</td><td>CircLigase II ssDNA Ligase</td></tr><tr><td>Centrifuge tube filters</td><td>Sigma-Aldrich</td><td>CLS8160-96EA</td><td>Costar Spin-X centrifuge tube filters</td></tr><tr><td>D5628-1G DIGITONIN CRYSTALLINE</td><td>Sigma-Aldrich</td><td>D5628-1G</td><td>For cell treatment</td></tr><tr><td>Dark Reader Transilluminator&amp;nbsp;</td><td>Clare Chemical Research</td><td>Dark Reader DR89X Transilluminator</td><td>Blue light transilluminator</td></tr><tr><td>DNA Gel Loading Dye (6x)</td><td>Life Technologies Holdings Pte Ltd</td><td>R0611</td><td>Required for agarose gel electroporation</td></tr><tr><td>Dulbecco&#039;s Modified Eagle Medium</td><td>Pan BioTech</td><td>P04-03500</td><td>For Hela cell culture</td></tr><tr><td>Streptavidin magnetic beads</td><td>Life Technologies Holdings Pte Ltd</td><td>65002</td><td>Dynabeads MyOne Streptavidin C1&amp;nbsp;</td></tr><tr><td>Magnetic stand for 15&amp;nbsp;mL tubes</td><td>Life Technologies Holdings Pte Ltd</td><td>12301D</td><td>DynaMag-15</td></tr><tr><td>Magnetic stand for 15&amp;nbsp;mL tubes</td><td>Life Technologies Holdings Pte Ltd</td><td>12321D</td><td>DynaMag-2</td></tr><tr><td>ThermoMixer</td><td>Eppendorf</td><td>5382 000.015</td><td>Eppendorf ThermoMixer&amp;nbsp;C</td></tr><tr><td>Biotinylated psoralen</td><td>Life Technologies Holdings Pte Ltd</td><td>29986</td><td>EZ-Link Psoralen-PEG3-Biotin</td></tr><tr><td>F8T5/BL&amp;nbsp;</td><td>Hitachi</td><td>F8T5/BL&amp;nbsp;</td><td>365 nm UV bulb</td></tr><tr><td>Fetal Bovine Serum</td><td>Life Technologies Holdings Pte Ltd</td><td>10270106&amp;nbsp;&amp;nbsp;</td><td>Components of Hela medium</td></tr><tr><td>Formamide</td><td>Promega</td><td>H5052&amp;nbsp;&amp;nbsp;</td><td>Component in hybridization buffer</td></tr><tr><td>G8T5</td><td>Sankyo-Denki</td><td>G8T5</td><td>254 nm UV bulb</td></tr><tr><td>Glycogen</td><td>Life Technologies Holdings Pte Ltd</td><td>10814010</td><td>Required for nucleic acid precipitation</td></tr><tr><td>Nanodrop</td><td>Life Technologies Holdings Pte Ltd</td><td>Nanodrop 2000</td><td>Spectrophotometer for nucleic acidquantification</td></tr><tr><td>Nuclease free water&amp;nbsp;</td><td>First BASE</td><td>BUF-1180-500ml&amp;nbsp;&amp;nbsp;</td><td></td></tr><tr><td>Penicillin Streptomycin&amp;nbsp;&amp;nbsp;</td><td>Life Technologies Holdings Pte Ltd</td><td>15140122&amp;nbsp;&amp;nbsp;</td><td>Components of Hela medium</td></tr><tr><td>High-Fidelity DNA polymerase (2x)</td><td>Life Technologies Holdings Pte Ltd</td><td>F531L&amp;nbsp;</td><td>Phusion High-Fidelity PCR Master Mix with HF Buffer (2x)</td></tr><tr><td>Primers Set 1</td><td>New England Biolabs</td><td>E7335L&amp;nbsp;</td><td>PCR primers</td></tr><tr><td>DNA Gel Extraction Kit</td><td>QIAgen</td><td>28106</td><td>QIAquick Gel Extraction Kit</td></tr><tr><td>Fluorometric Quantification kit</td><td>Life Technologies Holdings Pte Ltd</td><td>Q32854</td><td>Qubit dsDNA HS Assay Kit</td></tr><tr><td>RNA clean up kit</td><td>Qiagen</td><td>74106</td><td>RNeasy Mini Kit Silica-membrane column</td></tr><tr><td>Rnase Inhibitor</td><td>Life Technologies Holdings Pte Ltd</td><td>AM2696</td><td>SUPERase In</td></tr><tr><td>Reverse transcriptase</td><td>Life Technologies Holdings Pte Ltd</td><td>18080400</td><td>SuperScript III First-Strand Synthesis SuperMix</td></tr><tr><td>Nucleic Acid Gel Stain</td><td>Life Technologies Holdings Pte Ltd</td><td>S11494</td><td>SYBR GOLD NUCLEIC ACID 500 UL</td></tr><tr><td>T4 Polynucleotide Kinase</td><td>New England Biolabs</td><td>M0201L</td><td>End repair enzyme</td></tr><tr><td>T4 RNA Ligase 1</td><td>New England Biolabs</td><td>M0204L</td><td>Enzyme for proximity ligation</td></tr><tr><td>T4 RNA Ligase 2, truncated KQ</td><td>New England Biolabs</td><td>M0373L</td><td>Enzyme for adaptor ligation</td></tr><tr><td>Temed</td><td>Bio-Rad</td><td>1610801</td><td>TBE Urea gel component</td></tr><tr><td>Guanidinium thiocyanate-phenol-chloroform</td><td>Life Technologies Holdings Pte Ltd</td><td>15596018</td><td>TRIzol&amp;reg; Reagent for RNA extraction</td></tr><tr><td>Urea</td><td>First BASE</td><td>BIO-2070-5kg&amp;nbsp;&amp;nbsp;</td><td></td></tr><tr><td>UV crosslinker</td><td>Stratagene</td><td>400072</td><td>UV Stratalinker 1800</td></tr><tr><td>UV Transilluminator</td><td>UVP</td><td>95-0417-01</td><td>For visualizing of bands</td></tr><tr><td>Xylene Cyanol FF</td><td>Sigma-Aldrich</td><td>X4126-10G</td><td></td></tr><tr><td>DNA cleanup it</td><td>Zymo Research&amp;nbsp;</td><td>D4004</td><td>Zymo DNA concentrator-5&amp;nbsp;</td></tr><tr><td>&lt;strong&gt;List of software required&lt;/strong&gt;</td><td></td><td></td><td></td></tr><tr><td>FastQC software</td><td>0.11.4</td><td>http://www.bioinformatics.babraham.ac.uk/projects/fastqc/</td><td></td></tr><tr><td>SeqPrep software</td><td>1.0.7</td><td>https://github.com/jstjohn/SeqPrep</td><td></td></tr><tr><td>BWA software</td><td>0.7.12</td><td>http://bio-bwa.sourceforge.net/</td><td></td></tr><tr><td>SAMTOOLS software</td><td>1.2.1</td><td>http://www.htslib.org/</td><td></td></tr><tr><td>PULLSEQ software</td><td>1.0.2</td><td>https://github.com/bcthomas/pullseq</td><td></td></tr><tr><td>STAR software</td><td>2.5.0c</td><td>https://github.com/alexdobin/STAR</td><td></td></tr><tr><td>rem_dups.py PYTHON script</td><td>PYTHON 2.7.11</td><td>https://github.com/CSB5/splash/tree/master/src</td><td></td></tr><tr><td>find_chimeras.py PYTHON script</td><td>PYTHON 2.7.11</td><td>https://github.com/CSB5/splash/tree/master/src</td><td></td></tr><tr><td>pickJunctionReads.awk AWK script</td><td>AWK GNU 4.1.2</td><td>https://github.com/CSB5/splash/tree/master/src</td><td></td></tr><tr><td>&lt;strong&gt;Buffer composition&lt;/strong&gt;</td><td></td><td></td><td></td></tr><tr><td>&lt;strong&gt;Elution buffer&lt;/strong&gt;</td><td></td><td></td><td></td></tr><tr><td>0.3 M sodium acetate</td><td></td><td></td><td></td></tr><tr><td>&lt;strong&gt;Lysis Buffer&lt;/strong&gt;</td><td></td><td></td><td></td></tr><tr><td>50 mM Tris-Cl pH 7.0</td><td></td><td></td><td></td></tr><tr><td>10 mM EDTA</td><td></td><td></td><td></td></tr><tr><td>1% SDS</td><td></td><td></td><td></td></tr><tr><td>Always add Superase-in fresh before use except when washing beads</td><td></td><td></td><td></td></tr><tr><td>&lt;strong&gt;Proteinase K Buffer&amp;nbsp;&lt;/strong&gt;</td><td></td><td></td><td></td></tr><tr><td>100 mM NaCl</td><td></td><td></td><td></td></tr><tr><td>10 mM Tris-Cl pH 7.0</td><td></td><td></td><td></td></tr><tr><td>1 mM EDTA</td><td></td><td></td><td></td></tr><tr><td>0.5% SDS</td><td></td><td></td><td></td></tr><tr><td>&lt;strong&gt;Hybridization Buffer&lt;/strong&gt;</td><td></td><td></td><td></td></tr><tr><td>750 mM NaCl</td><td></td><td></td><td></td></tr><tr><td>1% SDS</td><td></td><td></td><td></td></tr><tr><td>50 mM Tris-Cl pH 7.0</td><td></td><td></td><td></td></tr><tr><td>1 mM EDTA</td><td></td><td></td><td></td></tr><tr><td>15% formamide (store in the dark at 4 &amp;deg;C)</td><td></td><td></td><td></td></tr><tr><td>Always add Superase-in fresh before use</td><td></td><td></td><td></td></tr><tr><td>&lt;strong&gt;2x SSC Wash Buffer&lt;/strong&gt;</td><td></td><td></td><td></td></tr><tr><td>2x NaCl and Sodium citrate (SSC) (diluted from 20x SSC Invitrogen stock)</td><td></td><td></td><td></td></tr><tr><td>0.5% SDS</td><td></td><td></td><td></td></tr><tr><td>&lt;strong&gt;2x RNA fragmentation buffer&lt;/strong&gt;</td><td></td><td></td><td></td></tr><tr><td>18 mM MgCl&lt;sub&gt;2&lt;/sub&gt;</td><td></td><td></td><td></td></tr><tr><td>450 mM KCl</td><td></td><td></td><td></td></tr><tr><td>300 mM Tris-Cl pH 8.3</td><td></td><td></td><td></td></tr><tr><td>&lt;strong&gt;2x RNA loading Dye&lt;/strong&gt;</td><td></td><td></td><td></td></tr><tr><td>95% Formamide</td><td></td><td></td><td></td></tr><tr><td>0.02% SDS</td><td></td><td></td><td></td></tr><tr><td>0.02% bromophenol blue</td><td></td><td></td><td></td></tr><tr><td>0.01% Xylene Cyanol</td><td></td><td></td><td></td></tr><tr><td>1 mM EDTA</td><td></td><td></td><td></td></tr><tr><td colspan="4">3&#039; RNA adapter sequence:&lt;br /&gt;
5rAppCTGTAGGCACCATCAAT/3ddC</td></tr><tr><td colspan="4">RT primer sequence:&lt;br /&gt;
AGATCGGAAGAGCGTCGTGTAGGGAAAGAGTGTAGATCTCGGTGGTCGC/iSp18/CACTCA/iSp18/TTCAGACGTGTGCTCTTCCGATCTATTGATGGTGCCTACAG</td></tr></tbody></table>

mapping rna-rna interactions globally using biotinylated psoralen

了解RNA与自身和其他人的相互作用是了解细胞中基于RNA的基因调控的关键。虽然已经显示RNA-RNA相互作用（例如微小RNA-mRNA相互作用）的实例调节基因表达，但是在细胞中发生RNA相互作用的完全程度仍然是未知的。以前研究RNA相互作用的方法主要集中在与特定蛋白质或RNA物种相互作用的RNA亚类。在这里，我们详细介绍了一种名为S等位基因的交联S型， L型化和S型选择性蛋白（SPLASH）的方法，其允许以无偏倚的方式在体内全基因组捕获RNA相互作用。 SPLASH利用体内交联，接近连接和高通量测序来鉴定全球范围内的分子内和分子间RNA碱基配对伙伴。 SPLASH可以应用于不同的生物体，包括细菌，酵母和人类细胞，以及a多样性的细胞条件，以促进理解不同细胞背景下RNA组织的动力学。整个实验SPLASH协议需要大约5天的时间才能完成，计算工作流程需要大约7天的时间才能完成。

图1描述了SPLASH工作流的原理图 。在0.01％的雌二醇和UV交联的情况下加入生物素化的补骨脂素后，从细胞中提取总RNA，并进行斑点印迹，以确保生物素化到RNA的交联有效发生（ 图2 ）。我们使用生物素化的20碱基寡核苷酸作为阳性对照，以滴定待添加到细胞中的生物素化补骨脂素的量，使得每150个碱基中?...

Watch this Scientific Journal Video about Mapping RNA-RNA Interactions Globally Using Biotinylated Psoralen at JoVE.com

Mapping RNA-RNA Interactions Globally Using Biotinylated Psoralen

在这里，我们详细介绍了P soralen交联，Ligated和S选择的H ybrids（SPLASH）的S等位基因的方法，使得能够在体内对分子内和分子间RNA-RNA相互作用进行基因组范围的映射。 SPLASH可用于研究包括酵母，细菌和人在内的生物体的RNA相互作用。

使用生物素化补骨脂素全球定位RNA-RNA相互作用

Knowing how RNAs interact with themselves and with others is key to understanding RNA based gene regulation in the cell. While examples of RNA-RNA ...

mapping-rna-rna-interactions-globally-using-biotinylated-psoralen

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Genetics

12.0K 次观看.  A*STAR. 在这里，我们详细介绍了P soralen交联，Ligated和S选择的H ybrids（SPLASH）的S等位基因的方法，使得能够在体内对分子内和分子间RNA-RNA相互作用进行基因组范围的映射。 SPLASH可用于研究包括酵母，细菌和人在内的生物体的RNA相互作用。 

视频: Mapping RNA-RNA Interactions Globally Using Biotinylated Psoralen

Novel RNA-Binding Proteins Isolation by the RaPID Methodology

RNA-binding proteins (RBPs) play important roles in every aspect of RNA metabolism and regulation. Their identification is a major challenge in modern biology. Only a few in vitro and in vivo methods enable the identification of RBPs associated with a particular target mRNA. However, their main limitations are the identification of RBPs in a non-cellular environment (in vitro) or the low efficiency isolation of RNA of interest (in vivo). An RNA-binding protein purification and identification (RaPID) methodology was designed to overcome these limitations in yeast and enable efficient isolation of proteins that are associated in vivo. To achieve this, the RNA of interest is tagged with MS2 loops, and co-expressed with a fusion protein of an MS2-binding protein and a streptavidin-binding protein (SBP). Cells are then subjected to crosslinking and lysed, and complexes are isolated through streptavidin beads. The proteins that co-purify with the tagged RNA can then be determined by mass spectrometry. We recently used this protocol to identify novel proteins associated with the ER-associated PMP1 mRNA. Here, we provide a detailed protocol of RaPID, and discuss some of its limitations and advantages.

RNA-protein interactions lie at the heart of many cellular processes. Here, we describe an in vivo method to isolate specific RNA and identify novel proteins that are associated with it. This could shed new light on how RNAs are regulated in the cell.

的快速方法新颖的RNA结合蛋白隔离

RNA-binding proteins (RBPs) play important roles in every aspect of RNA metabolism and regulation. Their identification is a major challenge in modern ...

科研

遗传学

Single Molecule Analysis of Laser Localized Psoralen Adducts

The DNA Damage Response (DDR) has been extensively characterized in studies of double strand breaks (DSBs) induced by laser micro beam irradiation in live cells. The DDR to helix distorting covalent DNA modifications, including interstrand DNA crosslinks (ICLs), is not as well defined. We have studied the DDR stimulated by ICLs, localized by laser photoactivation of immunotagged psoralens, in the nuclei of live cells. In order to address fundamental questions about adduct distribution and replication fork encounters, we combined laser localization with two other technologies. DNA fibers are often used to display the progress of replication forks by immunofluorescence of nucleoside analogues incorporated during short pulses. Immunoquantum dots have been widely employed for single molecule imaging. In the new approach, DNA fibers from cells carrying laser localized ICLs are spread onto microscope slides. The tagged ICLs are displayed with immunoquantum dots and the inter-lesion distances determined. Replication fork collisions with ICLs can be visualized and different encounter patterns identified and quantitated.

Lasers are frequently used in studies of the cellular response to DNA damage. However, they generate lesions whose spacing, frequency, and collisions with replication forks are rarely characterized. Here, we describe an approach that enables the determination of these parameters with laser localized interstrand crosslinks.

激光局部补骨脂素加合物的单分子分析

The DNA Damage Response (DDR) has been extensively characterized in studies of double strand breaks (DSBs) induced by laser micro beam irradiation in live ...

Sample Preparation for Mass Spectrometry-based Identification of RNA-binding Regions

Noncoding RNAs play important roles in several nuclear processes, including regulating gene expression, chromatin structure, and DNA repair. In most cases, the action of noncoding RNAs is mediated by proteins whose functions are in turn regulated by these interactions with noncoding RNAs. Consistent with this, a growing number of proteins involved in nuclear functions have been reported to bind RNA and in a few cases the RNA-binding regions of these proteins have been mapped, often through laborious, candidate-based methods.
Here, we report a detailed protocol to perform a high-throughput, proteome-wide unbiased identification of RNA-binding proteins and their RNA-binding regions. The methodology relies on the incorporation of a photoreactive uridine analog in the cellular RNA, followed by UV-mediated protein-RNA crosslinking, and mass spectrometry analyses to reveal RNA-crosslinked peptides within the proteome. Although we describe the procedure for mouse embryonic stem cells, the protocol should be easily adapted to a variety of cultured cells.

We describe a protocol to identify RNA-binding proteins and map their RNA-binding regions in live cells using UV-mediated photocrosslinking and mass spectrometry.

RNA 结合区质量谱鉴定的样品制备

Noncoding RNAs play important roles in several nuclear processes, including regulating gene expression, chromatin structure, and DNA repair. In most ...

生物化学

mRNA Interactome Capture from Plant Protoplasts

RNA-binding proteins (RBPs) determine the fates of RNAs. They participate in all RNA biogenesis pathways and especially contribute to post-transcriptional gene regulation (PTGR) of messenger RNAs (mRNAs). In the past few years, a number of mRNA-bound proteomes from yeast and mammalian cell lines have been successfully isolated through the use of a novel method called &#34;mRNA interactome capture,&#34; which allows for the identification of mRNA-binding proteins (mRBPs) directly from a physiological environment. The method is composed of in vivo ultraviolet (UV) crosslinking, pull-down and purification of messenger ribonucleoprotein complexes (mRNPs) by oligo(dT) beads, and the subsequent identification of the crosslinked proteins by mass spectrometry (MS). Very recently, by applying the same method, several plant mRNA-bound proteomes have been reported simultaneously from different Arabidopsis tissue sources: etiolated seedlings, leaf tissue, leaf mesophyll protoplasts, and cultured root cells. Here, we present the optimized mRNA interactome capture method for Arabidopsis thaliana leaf mesophyll protoplasts, a cell type that serves as a versatile tool for experiments that include various cellular assays. The conditions for optimal protein yield include the amount of starting tissue and the duration of UV irradiation. In the mRNA-bound proteome obtained from a medium-scale experiment (107 cells), RBPs noted to have RNA-binding capacity were found to be overrepresented, and many novel RBPs were identified. The experiment can be scaled up (109 cells), and the optimized method can be applied to other plant cell types and species to broadly isolate, catalog, and compare mRNA-bound proteomes in plants.

Here, we present an interactome capture protocol applied to Arabidopsis thaliana leaf mesophyll protoplasts. This method critically relies on in vivo UV crosslinking and allows for the isolation and identification of plant mRNA-binding proteins from a physiological environment.

mRNA从植物原生质体捕获

RNA-binding proteins (RBPs) determine the fates of RNAs. They participate in all RNA biogenesis pathways and especially contribute to post-transcriptional ...

Capture and Identification of RNA-binding Proteins by Using Click Chemistry-assisted RNA-interactome Capture (CARIC) Strategy

A comprehensive identification of RNA-binding proteins (RBPs) is key to understanding the posttranscriptional regulatory network in cells. A widely used strategy for RBP capture exploits the polyadenylation [poly(A)] of target RNAs, which mostly occurs on eukaryotic mature mRNAs, leaving most binding proteins of non-poly(A) RNAs unidentified. Here we describe the detailed procedures of a recently reported method termed click chemistry-assisted RNA-interactome capture (CARIC), which enables the transcriptome-wide capture of both poly(A) and non-poly(A) RBPs by combining the metabolic labeling of RNAs, in vivo UV cross-linking, and bioorthogonal tagging.

Capture and Identification of RNA-binding Proteins by Using Click Chemistry-assisted RNA-interactome Capture CARIC Strategy

A detailed protocol for applying the click chemistry-assisted RNA-interactome capture (CARIC) strategy to identify proteins binding to both coding and noncoding RNAs is presented.

利用单击化学辅助 rna-路特捕获 (无花果) 策略捕获和鉴定 rna 结合蛋白

A comprehensive identification of RNA-binding proteins (RBPs) is key to understanding the posttranscriptional regulatory network in cells. A widely used ...

In Vitro Biochemical Assays using Biotin Labels to Study Protein-Nucleic Acid Interactions

Protein-nucleic acid interactions play important roles in biological processes such as transcription, recombination, and RNA metabolism. Experimental methods to study protein-nucleic acid interactions require the use of fluorescent tags, radioactive isotopes, or other labels to detect and analyze specific target molecules. Biotin, a non-radioactive nucleic acid label, is commonly used in electrophoretic mobility shift assays (EMSA) but has not been regularly employed to monitor protein activity during nucleic acid processes. This protocol illustrates the utility of biotin labeling during in vitro enzymatic reactions, demonstrating that this label works well with a range of different biochemical assays. Specifically, in alignment with previous findings using radioisotope 32P-labeled substrates, it is confirmed via biotin-labeled EMSA that MEIOB (a protein specifically involved in the meiotic recombination) is a DNA-binding protein, that MOV10 (an RNA helicase) resolves biotin-labeled RNA duplex structures, and that MEIOB cleaves biotin-labeled single-stranded DNA. This study demonstrates that biotin is capable of substituting 32P in various nucleic acid-related biochemical assays in vitro.&#160;

Presented here are protocols for in vitro biochemical assays using biotin labels that may be widely&#160;applicable for studying protein-nucleic acid interactions.

使用生物素标签研究蛋白质-核酸相互作用的体外生化分析

Protein-nucleic acid interactions play important roles in biological processes such as transcription, recombination, and RNA metabolism. Experimental ...

Single-step Purification of Macromolecular Complexes Using RNA Attached to Biotin and a Photo-cleavable Linker

For many years, the exceptionally strong and rapidly formed interaction between biotin and streptavidin has been successfully utilized for partial purification of biologically important RNA/protein complexes. However, this strategy suffers from one major disadvantage that limits its broader utilization: the biotin/streptavidin interaction can be broken only under denaturing conditions that also disrupt the integrity of the eluted complexes, hence precluding their subsequent functional analysis and/or further purification by other methods. In addition, the eluted samples are frequently contaminated with the background proteins that nonspecifically associate with streptavidin beads, complicating the analysis of the purified complexes by silver staining and mass spectrometry. To overcome these limitations, we developed a variant of the biotin/streptavidin strategy in which biotin is attached to an RNA substrate via a photo-cleavable linker and the complexes immobilized on streptavidin beads are selectively eluted to solution in a native form by long wave UV, leaving the background proteins on the beads. Shorter RNA binding substrates can be synthesized chemically with biotin and the photo-cleavable linker covalently attached to the 5&#39; end of the RNA, whereas longer RNA substrates can be provided with the two groups by a complementary oligonucleotide. These two variants of the UV-elution method were tested for purification of the U7 snRNP-dependent processing complexes that cleave histone pre-mRNAs at the 3&#39; end and they both proved to compare favorably to other previously developed purification methods. The UV-eluted samples contained readily detectable amounts of the U7 snRNP that was free of major protein contaminants and suitable for direct analysis by mass spectrometry and functional assays. The described method can be readily adapted for purification of other RNA binding complexes and used in conjunction with single- and double-stranded DNA binding sites to purify DNA-specific proteins and macromolecular complexes.

RNA/protein complexes purified using botin-streptavidin strategy are eluted to solution under denaturing conditions in a form unsuitable for further purification and functional analysis. Here, we describe a modification of this strategy that utilizes a photo-cleavable linker in RNA and a gentle UV-elution step, yielding native and fully functional RNA/protein complexes.

生物素和光裂解链接器所附 rna 对高分子配合物的单步纯化

For many years, the exceptionally strong and rapidly formed interaction between biotin and streptavidin has been successfully utilized for partial ...

Identification of RNAs Engaged in Direct RNA-RNA Interaction with a Long Non-Coding RNA

The growing role attributed nowadays to long non-coding RNAs (lncRNA) in physiology and pathophysiology makes it crucial to characterize their interactome by identifying their molecular partners, DNA, proteins and/or RNAs. The latter can interact with lncRNA through networks involving proteins, but they can also be engaged in direct RNA/RNA interactions. We, therefore, developed an easy-to-use RNA pull-down procedure that allowed identification of RNAs engaged in direct RNA/RNA interaction with a lncRNA using psoralen, a molecule that cross-links only RNA/RNA interactions. Bioinformatics modeling of the lncRNA secondary structure allowed the selection of several specific antisense DNA oligonucleotide probes with a strong affinity for regions displaying a low probability of internal base pairing. Since the specific probes that were designed targeted accessible regions throughout the length of the lncRNA, the RNA-interaction zones could be delineated in the sequence of the lncRNA. When coupled with a high throughput RNA sequencing, this protocol can be used for the whole direct RNA interactome studies of a lncRNA of interest.

<meta charset="utf8"></head><body>鉴定与长链非编码 RNA 直接 RNA-RNA 相互作用的 RNA

An easy-to-use RNA pull-down protocol is designed for the identification of RNAs engaged in direct RNA/RNA interaction with a long non-coding RNA. The protocol uses psoralen as a fixative to cross-link only RNA/RNA interactions and provides the whole direct RNA interactome of a long non-coding RNA when coupled with RNA sequencing.

鉴定与长链非编码 RNA 直接 RNA-RNA 相互作用的 RNA

The growing role attributed nowadays to long non-coding RNAs (lncRNA) in physiology and pathophysiology makes it crucial to characterize their interactome ...

PAR-CliP - A Method to Identify Transcriptome-wide the Binding Sites of RNA Binding Proteins

RNA transcripts are subjected to post-transcriptional gene regulation by interacting with hundreds of RNA-binding proteins (RBPs) and microRNA-containing ribonucleoprotein complexes (miRNPs) that are often expressed in a cell-type dependently. To understand how the interplay of these RNA-binding factors affects the regulation of individual transcripts, high resolution maps of in vivo protein-RNA interactions are necessary1.
A combination of genetic, biochemical and computational approaches are typically applied to identify RNA-RBP or RNA-RNP interactions. Microarray profiling of RNAs associated with immunopurified RBPs (RIP-Chip)2 defines targets at a transcriptome level, but its application is limited to the characterization of kinetically stable interactions and only in rare cases3,4 allows to identify the RBP recognition element (RRE) within the long target RNA. More direct RBP target site information is obtained by combining in vivo UV crosslinking5,6 with immunoprecipitation7-9 followed by the isolation of crosslinked RNA segments and cDNA sequencing (CLIP)10. CLIP was used to identify targets of a number of RBPs11-17. However, CLIP is limited by the low efficiency of UV 254 nm RNA-protein crosslinking, and the location of the crosslink is not readily identifiable within the sequenced crosslinked fragments, making it difficult to separate UV-crosslinked target RNA segments from background non-crosslinked RNA fragments also present in the sample.
We developed a powerful cell-based crosslinking approach to determine at high resolution and transcriptome-wide the binding sites of cellular RBPs and miRNPs that we term PAR-CliP (Photoactivatable-Ribonucleoside-Enhanced Crosslinking and Immunoprecipitation) (see Fig. 1A for an outline of the method). The method relies on the incorporation of photoreactive ribonucleoside analogs, such as 4-thiouridine (4-SU) and 6-thioguanosine (6-SG) into nascent RNA transcripts by living cells. Irradiation of the cells by UV light of 365 nm induces efficient crosslinking of photoreactive nucleoside-labeled cellular RNAs to interacting RBPs. Immunoprecipitation of the RBP of interest is followed by isolation of the crosslinked and coimmunoprecipitated RNA. The isolated RNA is converted into a cDNA library and deep sequenced using Solexa technology. One characteristic feature of cDNA libraries prepared by PAR-CliP is that the precise position of crosslinking can be identified by mutations residing in the sequenced cDNA. When using 4-SU, crosslinked sequences thymidine to cytidine transition, whereas using 6-SG results in guanosine to adenosine mutations. The presence of the mutations in crosslinked sequences makes it possible to separate them from the background of sequences derived from abundant cellular RNAs.
Application of the method to a number of diverse RNA binding proteins was reported in Hafner et al.18

RNA transcripts are subject to extensive posttranscriptional regulation that is mediated by a multitude of trans-acting RNA-binding proteins (RBPs). Here we present a generalizable method to identify precisely and on a transcriptome-wide scale the RNA binding sites of RBPs.

PAR剪辑 - 一个方法来确定全转录组学，RNA结合蛋白的结合位点

RNA transcripts are subjected to post-transcriptional gene regulation by interacting with hundreds of RNA-binding proteins (RBPs) and microRNA-containing ...

使用生物素化补骨脂素全球定位RNA-RNA相互作用

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